Project Summary/Abstract This is a first-in-human, safety and feasibility study of bronchoscopic cryo-immunotherapy (BCI) in advanced non-small cell lung cancer (NSCLC). Cryo-immunotherapy involves the cryoablation of tumors as an in-situ vaccination, generating tumor-specific CD8+ T cell activation and proliferation, and has been demonstrated in both in syngeneic murine models of malignancy as well as in early-phase human clinical trials l [1]. Additionally, cryo-immunotherapy may synergize with other forms of immunotherapy ? such as immune checkpoint inhibitors (CPI) targeting the PD-1/PD-L1 pathway - to elicit enhanced anti-tumor immune responses and improved clinical outcomes in a variety of cancers, including NSCLC [2-6]. The combination of BCI with CPI may enable anti-tumor immune responses in patients who are refractory to therapy with CPI alone. Bronchoscopic cryoablation has been utilized for over twenty years for safe and effective palliative treatment and debulking of endobronchial tumors in the central airways, but has not yet been applied to the treatment of peripheral lung tumors [7, 8]. Percutaneous cryoablation has been utilized for more than a decade to treat peripheral primary and secondary lung tumors that are not amenable to surgical resection [9]. In percutaneous cryoablation, rigid cryotherapy probes are inserted into the target tumor via transthoracic computed tomography (CT) guidance, traversing the pleura and lung parenchyma with a resultant high rate of pneumothoraces [9] . In BCI, peripheral lung tumors will be accessed directly via the airway, minimizing the risk of pneumothoraces. BCI can also be performed during the course of standard of care bronchoscopic procedures, and potentially at much shorter procedure times than percutaneous cryoablation, The primary objectives of this study are to establish the safety and feasibility of BCI, as well as to determine the maximum tolerated dose - i.e. optimal duration of freeze time. The secondary objective of this study is to assess for BCI-induced anti-tumor immune responses by multiplex flow cytometry (FACS) analysis of pre- and post BCI cellular responses in peripheral blood, including post- BCI CD8+ T cell expression of PD-1 and markers of dendritic cell (DC) activation. Additional analysis of peripheral blood lymphocytes will be performed utilizing the PanCancer OncoImmuneTM profile panel (NanoString Technology, Seattle, WA) to assess for changes in lymphocyte gene expression. . We will be obtaining pre-BCI bronchoalveolar lavage (BAL) specimens from lung units both adjacent and distal to the tumor site to correlate findings in pre-BCI BAL lymphocytes and the strength of BCI induced anti-tumor immune responses. Finally, patients undergoing BCI will be followed longitudinally for changes in tumor size on chest CT scans using standard RECIST measurements[10] and also evaluated for progression-free and overall survival.